Investigation of the effect of low oxygen tension on the osteogenic differentiation of human mesenchymal stem cells

Osteogenic differentiation of hMSC into osteoblasts is a
prerequisite for subsequent bone formation. Numerous studies have
explored osteogenic differentiation under standard tissue culture
conditions, which usually employ 21% of oxygen. However, bone
precursor cells such as hMSC reside in stem cell niches of low
oxygen atmospheres. Furthermore, they are subjected to low oxygen
concentrations when cultured on three dimensional scaffolds in
vitro for bone tissue engineering purposes, and even more so after
transplantation when vascularisation has yet to be established.
Similarly, hMSC are exposed to low oxygen in the fracture
microenvironment following bony injury. Recent studies revealed
that hypoxic preconditioning improves cellular engraftment and
survival in low oxygen atmospheres. In the present study we
therefore investigated the osteogenic differentiation potential of
hMSC under 2% O2 (hypoxia) in comparison to a standard tissue
culture oxygen atmosphere of 21% (normoxia). The success of
differentiation was validated through Alizarin red staining and
RT-PCR analysis of osteoblast markers ALP and OPN. We assessed
osteogenic differentiation of hMSC following hypoxic
preconditioning to address whether this pretreatment is beneficial
for subsequent differentiation under low oxygen tension. To
validate our findings we carefully characterised the extent of
hypoxia exerted on cells with respect to cell survival (WST assay)
and proliferation (growth curve). Furthermore we also tried to
elucidate the role of HIF-1 alpha with respect to osteogenic
differentiation under hypoxia via silencing RNA and DFO, a
pharmacological agent. Finally we tested whether an immortalized
hMSC-line (SCP-1) would serve as a model system for hMSC. We found
that hMSC proliferate better if cultured under 2% of oxygen. We
confirmed that osteogenic differentiation of hMSC is indeed
inhibited under hypoxia. We showed for the first time that hypoxic
preconditioning of hMSC prior to osteogenic induction restores
osteogenic differentiation of hMSC under hypoxia. HIF-1 alpha
seemed not to play a significant role in osteogenic differentiation
under hypoxia, as transiently knocking down of HIF-1 alpha in
preconditioned samples did not show any differences in their
osteogenic differentiation. Moreover stabilising Hif-1 alpha in
hypoxic samples did not yield any osteogenic differentiation either
substantiating the notion that HIF-1 alpha does not have a direct
role in the osteogenic differentiation of hMSC under hypoxia.
Together our data suggest that hypoxia favours stemness over
differentiation by upregulating embryonic stem cell markers like
OCT-4 and NANOG. Hypoxic preconditioning may help to restore the
otherwise reduced osteogenic potential of hMSC, either within a
hypoxic fracture environment or at the site of implantation of
tissue engineered bone constructs. We therefore believe that
hypoxic preconditioning is a helpful tool for successful
regenerative cell-based therapies in bone tissue engineering. SCP-1
cells might be used as a model system for hMSC as they are easy to
handle, can be cultured to a desired cell number within a very
short period of time, are relatively inexpensive and above all do
not go into senescence as seen with hMSC after approximately 20
passages. Apart from their distinct advantages SCP-1 cells still
maintain the specific CD markers characteristic for hMSC and are
able to differentiate into adipogenic, osteogenic and chondrogenic
lineages. However for in vivo experiments in animals a constant
monitoring of neoplastic transformation is mandatory